CN113913374B - Serum-free culture method of umbilical cord mesenchymal stem cells - Google Patents
Serum-free culture method of umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a serum-free culture method of umbilical cord mesenchymal stem cells, which comprises the following steps: s1: culturing third generation mesenchymal stem cells and screening; s2: continuing culturing the selected third-generation cells; s3: adding an inducer in the culture process to convert into islet cells; the culture device comprises an incubator, a storage assembly, a control assembly and a protection plate, wherein the storage assembly is arranged in the incubator and comprises a placement frame, the control assembly comprises a control box, a control bin is arranged on the front surface of the control box, a control port is arranged on one side of the top of the control bin, and a control mechanism is arranged in the control port. The beneficial effects of the invention are as follows: the invention provides the mesenchymal stem cells extracted from the umbilical cord and the preparation method thereof, which have the advantages of easily available material sources, simple, convenient and efficient operation, can effectively reduce the culture cost of MSCs and obtain a large number of MSC cells, and provides a foundation for clinical application of the MSCs.
Description
Technical Field
The invention relates to a culture method of umbilical cord mesenchymal stem cells, in particular to a serum-free culture method of umbilical cord mesenchymal stem cells, and belongs to the technical field of stem cell culture.
Background
Mesenchymal stem cells (Mesenchymal stem cells, MSCs) are adult stem cells with multidirectional differentiation potential, and the stem cells can be self-replicated, self-renew, stimulate tissue growth and repair, have low pollution sources, are convenient to obtain, have stable biological performance and are more original in immunogenicity. Under the proper in vitro experimental conditions, the amplification and the preservation are easy. The mesenchymal stem cells proliferate immune cells in vivo by changing cytokines secreted by T lymphocytes, B lymphocytes, natural killer cells and dendritic cells, so that the mesenchymal stem cells have extremely wide application prospects in the field of cell therapy.
Along with the continuous progress of scientific technology, the search of new and more potential mesenchymal stem cells has become a research hotspot nowadays, and the mesenchymal stem cells can promote tissue modification and improve immunity and have obvious curative effects on autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and the like clinically. How to obtain a sufficient number of mesenchymal stem cells with higher potential is an important guarantee of the application of MSCs in clinic, so as to further widen the understanding of biological properties of umbilical mesenchymal stem cells and provide support for the clinical application of the mesenchymal stem cells.
The existing cell culture device is mainly carried out by adopting an incubator, so that cells can be conveniently cultured, the shape of the incubator for some experiments is similar to that of a small refrigerator, the incubator is quite light, the incubator is similar to that of a refrigerator in a sampling mode of a cultured sample, a sealing door is opened for direct taking, a layer of sealing plate is only added for some of protection, a protection plate is also directly opened when partial samples are taken and placed, and all samples are exposed, so that the subsequent culture result can be influenced.
Disclosure of Invention
The invention aims to solve the problems and provide a serum-free culture method of umbilical cord mesenchymal stem cells.
The invention realizes the aim through the following technical scheme, and the serum-free culture method of the umbilical cord mesenchymal stem cells comprises the following steps:
s1: culturing third generation mesenchymal stem cells by using a serum-free culture medium, and carrying out third generation cell screening;
s2: placing the selected third-generation cells into a culture medium containing islet proteins for continuous culture;
s3: and adding an inducer in the culture process to induce the mesenchymal stem cells to be converted into islet cells, and screening and removing untransformed cells.
Preferably, the serum-free culture medium comprises the following components in percentage by mass: 1-20ng/mL of fibroblast growth factor, 10-45ng/mL of epidermal growth factor, 1-8mg/mL of human insulin, 5-15ng/mL of fibronectin, 5-10mg/mL of vitamin, 5-10mg/mL of transforming growth factor, 10-100mg/mL of human serum albumin, 50-200 mu g/mL of fibronectin and 80-100ug/mL of penicillin;
the screening of the third generation cells comprises the following processes;
process one: firstly, taking out a culture medium in which third-generation cells are cultured from a culture device;
and a second process: extracting the basal liquid in the culture medium, and then pumping the third-generation cells into an activity detection reagent;
and a third process: and (3) carrying out a process under a microscope, screening out third-generation cells with stronger activity, and then placing the selected cells in a new serum-free culture medium again for proliferation culture.
Preferably, the inducer comprises the following components in percentage by mass: 5-25mg/mL of niacinamide, 15-45mg/mL of islet protein, 20-60mg/mL of nerve growth factor, 5-10mg/mL of activin, 5-10mg/mL of transferrin, 5-50ng/mL of insulin-like growth factor and 1-10mg/mL of recombinant human insulin.
Preferably, the culture apparatus used in S1 includes:
the front surface of the incubator is provided with a sealing door;
the storage assembly is arranged in the incubator and comprises a placement rack, a plurality of placement bins are arranged on the front face of the placement rack, support pads are arranged on the inner walls of the bottom ends of the placement bins, and a baffle is arranged on the back face of the placement rack;
the control assembly is arranged on the back of the baffle plate, the control assembly comprises a control box, a control bin is arranged on the front of the control box, two moving ports are arranged on the inner wall of the bottom end of the control bin, a first moving plate is connected in one moving port in a sliding and inserting mode, a second moving plate is connected in the other moving port in a sliding and inserting mode, one side of the top of the control bin is positioned on the front of the control bin, and a control port is arranged in the control port;
the protection plates are arranged on the front face of the placing frame and are hinged to the bin openings on the front face of the placing bin respectively.
Preferably, the front of incubator is provided with the display screen, the front of closing door is provided with the handle, the cultivation storehouse has been seted up to the front of incubator, and be located one side of cultivation storehouse and seted up the control hole.
Preferably, the front of the placing rack is provided with a plurality of penetrating holes corresponding to a plurality of placing bins, the inner walls of two sides of the penetrating holes are provided with locking holes, two adjacent locking holes are communicated with each other, the inner walls of two sides of the placing bins are provided with vent holes, the vent holes of two adjacent sides are communicated with each other, and one side of the top of the placing rack is provided with a sliding opening.
Preferably, the front of first movable plate and second movable plate all fixedly connected with a plurality of shutoff boards, fixedly connected with gangbar between first movable plate and the second movable plate bottom, one side of second movable plate is provided with a plurality of teeth.
Preferably, the control mechanism comprises a control rod, the control rod is connected with the control hole in a rotating and penetrating way, a first control fluted disc and a second control fluted disc are respectively sleeved on a lever arm of the control rod, and the first control fluted disc is meshed with the plurality of teeth.
Preferably, the outer wall meshing of second control fluted disc bottom has the rack board, rack board and sliding gate slip alternate connection, the top and the bottom of rack board all are provided with a plurality of connecting strips, a plurality of the equal fixedly connected with locking lever of one side of connecting strip, a plurality of the locking lever respectively with a plurality of locking hole slip alternate connection.
Preferably, the bottom of guard plate back fixedly connected with alternates the post, it alternates with one of them alternate hole slip and connects to wear to insert the post, it has offered the fixed orifices to wear to insert one side of post, the fixed orifices alternates with one of them locking lever slip and connects, the positive bottom fixedly connected with of guard plate draws the piece.
The beneficial effects of the invention are as follows:
the invention provides the mesenchymal stem cells extracted from the umbilical cord and the preparation method thereof, which have the advantages of easily available material sources, simple, convenient and efficient operation, can effectively reduce the culture cost of MSCs and obtain a large number of MSC cells, and provides a foundation for clinical application of the MSCs.
Secondly, the culture device of the invention is designed on the storage component and then is controlled by matching with the control component, so that the opening and closing states of the storage bins are relatively independent when samples are taken and placed, and the closing and opening of each storage bin are flexibly regulated by the rotation regulation of the control component, thereby ensuring that other samples in culture are not influenced when the samples are taken.
Thirdly, the culture device is driven by rotating the control rod and utilizing the two control fluted discs to respectively carry out meshing transmission, and then the two movable plates and the rack plate are matched for linkage, so that when a sample needs to be taken out, the two movable plates drive the plugging plate to separate the placing bins independently, and meanwhile, the rack plate drives the locking rod to unlock, so that opposite sampling operation can be realized.
Drawings
FIG. 1 is a schematic view showing the overall structure of a culture apparatus according to the present invention;
FIG. 2 is a schematic illustration of the connection of the incubator and storage assembly of FIG. 1 in accordance with the present invention;
FIG. 3 is a schematic view of the connection structure of the rack and the control assembly of FIG. 2 according to the present invention;
FIG. 4 is a schematic view of a rack according to the present invention;
FIG. 5 is an enlarged schematic view of the structure of FIG. 4A according to the present invention;
FIG. 6 is a schematic diagram of a control assembly according to the present invention;
FIG. 7 is an enlarged schematic view of the structure of FIG. 6B in accordance with the present invention;
FIG. 8 is a schematic diagram of a control mechanism according to the present invention;
fig. 9 is a schematic structural view of the protection plate of the present invention.
In the figure: 1. an incubator; 101. closing the door; 2. a placing rack; 201. a baffle; 202. a support pad; 203. penetrating the jack; 204. a vent hole; 3. a control box; 301. a first moving plate; 302. a second moving plate; 303. a plugging plate; 304. a linkage rod; 305. teeth; 4. a control lever; 401. a first control toothed disc; 402. a second control fluted disc; 5. rack plate; 501. a connecting strip; 502. a locking lever; 6. a protection plate; 601. the column is inserted.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1-9, a serum-free culture method of umbilical cord mesenchymal stem cells comprises the following steps:
s1: culturing third generation mesenchymal stem cells by using a serum-free culture medium, and carrying out third generation cell screening;
the serum-free culture medium comprises the following components in percentage by mass: 1-20ng/mL of fibroblast growth factor, 10-45ng/mL of epidermal growth factor, 1-8mg/mL of human insulin, 5-15ng/mL of fibronectin, 5-10mg/mL of vitamin, 5-10mg/mL of transforming growth factor, 10-100mg/mL of human serum albumin, 50-200 mu g/mL of fibronectin and 80-100ug/mL of penicillin;
epidermal Growth Factor (EGF) is a thermostable single-chain low molecular polypeptide consisting of 53 amino acid residues. EGF specifically recognizes and binds to EGF receptor on target cells, and then undergoes a series of biochemical reactions, which finally promote DNA synthesis and mitosis of target cells. EGF has no glycosyl part, is very stable, and is heat-resistant and acid-resistant;
transforming Growth Factor (TGF) refers to two classes of polypeptide growth factors, transforming growth factor-alpha and transforming growth factor-beta. Transforming growth factor-alpha is produced by macrophages, brain cells and epidermal cells, and induces cellular development.
The screening of the third generation cells comprises the following processes;
process one: firstly, taking out a culture medium in which third-generation cells are cultured from a culture device;
and a second process: extracting the basal liquid in the culture medium, and then pumping the third-generation cells into an activity detection reagent;
and a third process: and (3) carrying out a process under a microscope, screening out third-generation cells with stronger activity, and then placing the selected cells in a new serum-free culture medium again for proliferation culture.
S2: placing the selected third-generation cells into a culture medium containing islet proteins for continuous culture;
s3: adding an inducer in the culture process, inducing the mesenchymal stem cells to be converted into islet cells, and screening and removing untransformed cells;
the inducer comprises the following components in percentage by mass: 5-25mg/mL of niacinamide, 15-45mg/mL of islet protein, 20-60mg/mL of nerve growth factor, 5-10mg/mL of activin, 5-10mg/mL of transferrin, 5-50ng/mL of insulin-like growth factor and 1-10mg/mL of recombinant human insulin.
The culture apparatus used in S1 includes:
incubator 1, the front of incubator 1 is provided with a closing door 101;
incubator 1 is a carbon dioxide incubator of the prior art, can provide environmental conditions required for cell survival, and can be sealed well by sealing door 101.
As a technical optimization scheme of the invention, the front surface of the incubator 1 is provided with a display screen, the front surface of the sealing door 101 is provided with a handle, the front surface of the incubator 1 is provided with a culture bin, the front surface of the incubator 1 is provided with a control hole at one side of the culture bin;
the display screen can only display the environmental condition parameters in the incubator 1, and can set and modify the corresponding condition parameters through the touch screen keys.
The storage assembly is arranged in the incubator 1 and comprises a placement frame 2, a plurality of placement bins are arranged on the front face of the placement frame 2, support pads 202 are arranged on the inner walls of the bottom ends of the placement bins, and a baffle 201 is arranged on the back face of the placement frame 2;
the back of rack 2 is for being located the lateral wall that places the storehouse for the open design to the individual, and rack 2 is hollow structure simultaneously, conveniently provides control assembly's activity space, and baffle 201 carries out half protection shutoff to the back of rack 2.
As a technical optimization scheme of the invention, the front surface of the placement frame 2 is provided with a plurality of through holes 203 corresponding to a plurality of placement bins, the inner walls of two sides of the plurality of through holes 203 are provided with locking holes, two adjacent locking holes are communicated with each other, the inner walls of two sides of the plurality of placement bins are provided with vent holes 204, the vent holes 204 of two adjacent sides are communicated with each other, and one side of the top of the placement frame 2 is provided with a sliding opening;
the through holes 203 are correspondingly formed in the centers of the outer walls of the bottom of the placing bins, the vent holes 204 are formed in the inner walls of the two sides of the top of the placing bins, the bottom of each placing bin is ensured to be in a closed and independent state, the vent holes 204 are used for circulating culture gas provided by the incubator 1, and the sliding ports are communicated with the middle of the placing rack 2.
The control assembly is arranged on the back of the baffle 201 and comprises a control box 3, a control bin is arranged on the front of the control box 3, two moving ports are arranged on the inner wall of the bottom end of the control bin, a first moving plate 301 is connected in one moving port in a sliding and inserting mode, a second moving plate 302 is connected in the other moving port in a sliding and inserting mode, one side of the top of the control bin is provided with a control port on the front, and a control mechanism is arranged in the control port;
the opening width and length of the two moving ports are correspondingly the same as the width and thickness of the first moving plate 301 and the second moving plate 302, and the contact surface is smooth, so that the first moving plate 301 and the second moving plate 302 can well move in the vertical direction.
As a technical optimization scheme of the invention, the front surfaces of a first movable plate 301 and a second movable plate 302 are fixedly connected with a plurality of plugging plates 303, a linkage rod 304 is fixedly connected between the bottoms of the first movable plate 301 and the second movable plate 302, and a plurality of teeth 305 are arranged on one side of the second movable plate 302;
the plurality of teeth 305 are vertically and centrally symmetrically arranged at one side of the second moving plate 302 and near the rear surface.
As a technical optimization scheme of the invention, the control mechanism comprises a control rod 4, the control rod 4 is connected with a control hole in a rotating and penetrating way, a lever arm of the control rod 4 is respectively sleeved with a first control fluted disc 401 and a second control fluted disc 402, and the first control fluted disc 401 is meshed with a plurality of tooth teeth 305;
the first control fluted disc 401 rotates in the control port, and one end of the control rod 4 is connected with the inner wall of the back of the control port in a rotating and penetrating way.
The outer wall of the bottom end of the second control fluted disc 402 is meshed with a rack plate 5, the rack plate 5 is connected with a sliding port in a sliding penetrating way, a plurality of connecting strips 501 are arranged at the top end and the bottom end of the rack plate 5, locking rods 502 are fixedly connected to one sides of the connecting strips 501, and the locking rods 502 are respectively connected with a plurality of locking holes in a sliding penetrating way;
the locking bar 502 is arranged in a hemispherical shape at one end far away from the connecting bar 501, so that the locking bar can be more conveniently inserted into the locking hole and the fixing hole in a sliding manner.
The protection plates 6 are arranged on the front face of the placing frame 2, and are respectively hinged to the bin openings on the front faces of the placing bins;
as a technical optimization scheme of the invention, a penetrating column 601 is fixedly connected to the bottom of the back surface of the protection plate 6, the penetrating column 601 is in sliding penetrating connection with one of the penetrating holes 203, one side of the penetrating column 601 is provided with a fixing hole, the fixing hole is in sliding penetrating connection with one of the locking rods 502, and the bottom of the front surface of the protection plate 6 is fixedly connected with a pull block.
When the culture device is used, referring to fig. 1 to 9, in the process of cell culture, a culture medium is required to be stored;
performing a first operation, firstly opening the closing door 101, and then rotating the control rod 4 clockwise, wherein the control rod 4 drives the first control fluted disc 401 and the second control fluted disc 402 to synchronously rotate, and the following process can occur;
process one: the first control fluted disc 401 rotates clockwise, and the second movable plate 302 moves upwards through the meshing transmission with the tooth 305, the second movable plate 302 drives the first movable plate 301 to move upwards synchronously through the linkage rod 304, meanwhile, the first movable plate 301 and the second movable plate 302 drive the plugging plate 303 connected with the front surface to move upwards, the plugging plate 303 moves upwards to be blocked between two sides of adjacent placing bins, and adjacent vent holes 204 are plugged, and at the moment, the placing bins are in a relatively independent state;
and a second process: the second control fluted disc 402 rotates clockwise, and makes the rack board 5 move horizontally and transversely towards the direction close to the rack 2 through meshing transmission, at this time, the connecting strip 501 moves along with the rack board 5, and simultaneously drives the locking rod 502 to move synchronously, at this time, the locking rod 502 slides out from the fixing hole and is scaled into the locking hole, and the locking of the penetrating and inserting column 601 is released.
The second step is performed, the pull block is pinched to turn over the protection plate 6 upwards, and the penetrating column 601 slides out of the insertion hole 203 along with the protection plate 6 in the reverse rotation process.
And a third step of operation is carried out, the culture medium to be stored and cultivated is placed in a placing bin of the protection plate 6, meanwhile, for stability, the culture medium can be embedded in the supporting pad 202, after the placing is completed, the protection plate 6 is turned down, and the penetrating column 601 is penetrated into the penetrating hole 203.
A fourth step of operation is implemented, the control rod 4 is rotated anticlockwise, the control rod 4 drives the first control fluted disc 401 and the second control fluted disc 402 to rotate anticlockwise, at the moment, the first moving plate 301 and the second moving plate 302 drive the plugging plate 303 to move downwards under the meshing transmission, the plugging plate 303 releases the plugging of the vent hole 204, and all the placing bins are communicated with gas;
the rack plate 5 drives the connecting strip 501 to move away from the placing frame 2, and meanwhile the locking rod 502 moves along with the connecting strip 501 from the locking hole and slides to penetrate into the fixing hole, and the locking rod 502 cooperates with the fixing hole to lock the penetrating and inserting column 601 in the penetrating hole 203, so that the sealing of the protection plate 6 is completed.
And a fifth step of closing the sealing door 101, starting the incubator 1, and if the culture medium in the corresponding storage bin needs to be taken out, opening the protection plate 6 at the corresponding position after the first step and the second step of operation.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (3)
1. A serum-free culture method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps: the method comprises the following steps:
s1: a serum-free culture medium is used, and the culture medium is put into a culture device to culture third-generation mesenchymal stem cells, and the third-generation cell screening is performed by a screen;
s2: placing the selected third-generation cells into a culture medium containing islet proteins for continuous culture;
s3: adding an inducer in the culture process, inducing the mesenchymal stem cells to be converted into islet cells, and screening and removing untransformed cells;
wherein, the culture device used in step S1 comprises:
the incubator comprises an incubator (1), wherein a sealing door (101) is arranged on the front surface of the incubator (1);
the storage assembly is arranged in the incubator (1), the storage assembly comprises a placement rack (2), a plurality of placement bins are arranged on the front face of the placement rack (2), support pads (202) are arranged on the inner walls of the bottom ends of the placement bins, and a baffle (201) is arranged on the back face of the placement rack (2);
the control assembly is arranged on the back of the baffle plate (201), the control assembly comprises a control box (3), a control bin is arranged on the front of the control box (3), two moving ports are arranged on the inner wall of the bottom end of the control bin, a first moving plate (301) is connected in one moving port in a sliding and inserting mode, a second moving plate (302) is connected in the other moving port in a sliding and inserting mode, one side of the top of the control bin is positioned on the front of the control bin, a control port is arranged on the front of the control bin, and a control mechanism is arranged in the control port;
the protection plates (6) are arranged on the front surfaces of the placing frames (2) and are respectively hinged to the bin openings on the front surfaces of the placing bins;
the front of the incubator (1) is provided with a display screen, the front of the sealing door (101) is provided with a handle, the front of the incubator (1) is provided with a culture bin, and the front of the incubator (1) is provided with a control hole at one side of the culture bin;
the front surface of the placement frame (2) is provided with a plurality of penetrating holes (203) corresponding to a plurality of placement bins, the inner walls of two sides of the penetrating holes (203) are provided with locking holes, two adjacent locking holes are communicated with each other, the inner walls of two sides of the placement bins are provided with vent holes (204), the vent holes (204) of two adjacent sides are communicated with each other, and one side of the top of the placement frame (2) is provided with a sliding opening;
the front sides of the first moving plate (301) and the second moving plate (302) are fixedly connected with a plurality of plugging plates (303), a linkage rod (304) is fixedly connected between the bottoms of the first moving plate (301) and the second moving plate (302), and a plurality of teeth (305) are arranged on one side of the second moving plate (302);
the control mechanism comprises a control rod (4), the control rod (4) is connected with the control hole in a rotating and penetrating way, a first control fluted disc (401) and a second control fluted disc (402) are sleeved on a lever arm of the control rod (4), and the first control fluted disc (401) is meshed with a plurality of teeth (305);
the outer wall of the bottom end of the second control fluted disc (402) is meshed with a rack plate (5), the rack plate (5) is connected with a sliding port in a sliding penetrating mode, a plurality of connecting strips (501) are arranged at the top end and the bottom end of the rack plate (5), locking rods (502) are fixedly connected to one sides of the connecting strips (501), and the locking rods (502) are respectively connected with a plurality of locking holes in a sliding penetrating mode;
the bottom of guard plate (6) back fixedly connected with alternates post (601), alternate post (601) and one of them jack (203) slip interlude and connect, the fixed orifices has been seted up to one side of alternates post (601), fixed orifices and one of them locking lever (502) slip interlude and connect, the positive bottom fixedly connected with of guard plate (6) draws the piece.
2. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the steps of: the serum-free culture medium comprises the following components in percentage by mass: 1-20ng/mL of fibroblast growth factor, 10-45ng/mL of epidermal growth factor, 1-8mg/mL of human insulin, 5-15ng/mL of fibronectin, 5-10mg/mL of vitamin, 5-10mg/mL of transforming growth factor, 10-100mg/mL of human serum albumin, 50-200 mu g/mL of fibronectin and 80-100ug/mL of penicillin;
the screening of the third generation cells comprises the following processes;
process one: firstly, taking out a culture medium in which third-generation cells are cultured from a culture device;
and a second process: extracting the basal liquid in the culture medium, and then pumping the third-generation cells into an activity detection reagent;
and a third process: and (3) carrying out a process under a microscope, screening out third-generation cells with stronger activity, and then placing the selected cells in a new serum-free culture medium again for proliferation culture.
3. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 1, wherein the method comprises the steps of: the inducer comprises the following components in percentage by mass: 5-25mg/mL of niacinamide, 15-45mg/mL of islet protein, 20-60mg/mL of nerve growth factor, 5-10mg/mL of activin, 5-10mg/mL of transferrin, 5-50ng/mL of insulin-like growth factor and 1-10mg/mL of recombinant human insulin.
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